Separating T Cell Targeting Components onto Magnetically Clustered Nanoparticles Boosts Activation

T cell activation requires the coordination of a variety of signaling molecules including T cell receptor-specific signals and costimulatory signals. Altering the composition and distribution of costimulatory molecules during stimulation greatly affects T cell functionality for applications such as adoptive cell therapy (ACT), but the large diversity in these molecules complicates these studies. Here, we develop and validate a reductionist T cell activation platform that enables streamlined customization of stimulatory conditions. This platform is useful for the optimization of ACT protocols as well as the more general study of immune T cell activation. Rather than decorating particles with both signal 1 antigen and signal 2 costimulus, we use distinct, monospecific, paramagnetic nanoparticles, which are then clustered on the cell surface by a magnetic field. This allows for rapid synthesis and characterization of a small number of single-signal nanoparticles which can be systematically combined to explore and optimize T cell activation. By increasing cognate T cell enrichment and incorporating additional costimulatory molecules using this platform, we find significantly higher frequencies and numbers of cognate T cells stimulated from an endogenous population. The magnetic field-induced association of separate particles thus provides a tool for optimizing T cell activation for adoptive immunotherapy and other immunological studies

Biologically Inspired Design of Nanoparticle Artificial Antigen-Presenting Cells for Immunomodulation

Particles engineered to engage and interact with cell surface ligands and to modulate cells can be harnessed to explore basic biological questions as well as to devise cellular therapies. Biology has inspired the design of these particles, such as artificial antigen-presenting cells (aAPCs) for use in immunotherapy. While much has been learned about mimicking antigen presenting cell biology, as we decrease the size of aAPCs to the nanometer scale, we need to extend biomimetic design to include considerations of T cell biologyincluding T-cell receptor (TCR) organization. Here we describe the first quantitative analysis of particle size effect on aAPCs with both Signals 1 and 2 based on T cell biology. We show that aAPCs, larger than 300 nm, activate T cells more efficiently than smaller aAPCs, 50 nm. The 50 nm aAPCs require saturating doses or require artificial magnetic clustering to activate T cells. Increasing ligand density alone on the 50 nm aAPCs did not increase their ability to stimulate CD8+ T cells, confirming the size-dependent phenomenon. These data support the need for multireceptor ligation and activation of T-cell receptor (TCR) nanoclusters of similar sizes to 300 nm aAPCs. Quantitative analysis and modeling of a nanoparticle system provides insight into engineering constraints of aAPCs for T cell immunotherapy applications and offers a case study for other cell-modulating particles

Dual Targeting Nanoparticle Stimulates the Immune System To Inhibit Tumor Growth

We describe the development of a nanoparticle platform that overcomes the immunosuppressive tumor microenvironment. These nanoparticles are coated with two different antibodies that simultaneously block the inhibitory checkpoint PD-L1 signal and stimulate T cells <i>via</i> the 4-1BB co-stimulatory pathway. These “immunoswitch” particles significantly delay tumor growth and extend survival in multiple <i>in vivo</i> models of murine melanoma and colon cancer in comparison to the use of soluble antibodies or nanoparticles separately conjugated with the inhibitory and stimulating antibodies. Immunoswitch particles enhance effector-target cell conjugation and bypass the requirement for <i>a priori</i> knowledge of tumor antigens. The use of the immunoswitch nanoparticles resulted in an increased density, specificity, and <i>in vivo</i> functionality of tumor-infiltrating CD8+ T cells. Changes in the T cell receptor repertoire against a single tumor antigen indicate immunoswitch particles expand an effective set of T cell clones. Our data show the potential of a signal-switching approach to cancer immunotherapy that simultaneously targets two stages of the cancer immunity cycle resulting in robust antitumor activity

Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion <i>in vitro</i> and after adoptive transfer <i>in vivo</i>. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy

DN T cells have a unique transcript profile.

<p>(A) Comparison of the transcript profiles of DN T cells and autologous SP T cells (combined CD4 and CD8 T cells) isolated from inguinal and axillary lymph nodes of gld mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003465#s4" target="_blank">Materials and Methods</a>. The insets show purity and hallmarks properties of DN T cells including expression of TCR and B220 and lack of CD4 and CD8 on their surface. The Minus versus Average (MvA) plot shows intensity log-ratio M = log₂ (DN/SP) versus mean log-intensity A = [(log₂ (DN)+log₂ (SP)]/2. Out of 32,000 transcripts examined, expression of 160 genes was at least 3-fold higher in DN than in SP T cells. Highly expressed genes including junctional adhesion molecules, hydrolyase enzymes, and apoptotic death molecules are highlighted. Selected under-expressed genes in DN T cells relative to SP T cells are also highlighted. (B) Validation of expression of selected genes by real-time PCR. Expression level of each gene was normalized relative to expression of 18 s rRNA in the same cell subset. X-axis shows -fold change in expression of indicated genes in DN T cells relative to SP T cells. (C) Validation of specific expression of sdc1 by flow cytometry. Splenocytes were isolated from 16-week-old C3H-gld/gld mice, stained with APC-TCRβ, PerCP-CD4, FITC-CD8α, and PE-sdc1 or with APC-TCRβ, PerCP-CD4, FITC-CD8α, and PE-B220 mAbs and analyzed by FACS. Dot plot: TCR⁺ cells were gated followed by specific gating of DN (R5), CD4⁺ (R3), and CD8⁺ (R4) subsets. Histograms: Overlays show relative expression of TCR, B220, and sdc1 by gated CD4, CD8, and DN subsets.</p

Rapid proliferation of intestinal but not peripheral DN T cells in the steady state.

<p>Wild-type and gld mice (12-week-old) received two i.p. injections of BrdU during a 24-h period as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003465#s4" target="_blank">Materials and Methods</a>. Peripheral T cells and IEL were isolated and stained with APC-TCRβ, PerCP-CD4/PerCP-CD8α, FITC-BrdU and PE- sdc1 specific antibodies. TCR⁺ cells were gated and percentage of BrdU^high in the DN or SP (CD4⁺+CD8⁺) subsets in wt (A) and gld (B) mice were determined. The graph below each panel shows mean±SEM from two independent experiments with 2–3 mice per each genotype. *, P<0.05; N.S., not significantly different.</p

Phenotypic similarities of peripheral and intestinal DN T cells.

<p>(A) Single cell suspensions from spleens of gld mice or intestine (small and large intestines were combined) of gld or wt mice were stained for APC-TCRβ, PerCP-CD4, and PerCP-CD8α, and FITC-CD2, CD5 or B220. TCR⁺ cells were gated and expression of indicated molecules by DN and SP subsets were determined. Spleens of wt mice are not included in the analysis because of the paucity of DN T cells. (B) Similar distribution of Vβ expression by DN T cells. Peripheral T cells and IEL were isolated from 12-week-old C3H-gld/gld mice and surface-stained for TCR, CD4, CD8, and Vβ6, Vβ8, Vβ13 or Vβ14. After gating on TCR⁺ cells, the frequencies of different Vβs among DN, CD4 or CD8 T cells were determined. Data show mean±SD from one of two independent experiments. (C) Downregulation of sdc1 by TCR activation. Top panel: Analysis of TCR (left dot plot); and CD4 and CD8α expression (right dot plot) by freshly isolated DN cells prior to culture. The isolated cells were stained with TCR, CD4 and CD8 specific antibodies and their purity assessed by flow cytometry (>95% of isolated cells expressed TCR (left dot plot) and lacked CD4 and CD8 expression (middle dot plot). The histogram shows CFSE intensity in DN T cells before (day 0) and after (day 4) TCR activation. Bottom panel: Kinetics of sdc1 and CD62L downregulation by DN T cells in response to CD3/CD28 stimulation. Data from one of two independent experiments are shown.</p

Sdc1⁺ B220+ DN T cells accumulate in an age-dependent manner in the gut epithelium of wt mice with intact Fas pathway.

<p>T cells were isolated from the periphery and gut epithelium of wt or gld C3H mice of different ages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003465#s4" target="_blank">Materials and Methods</a>. Isolated cells from lymph nodes (LN), spleen, small intestine (SI), and large intestines were stained with APC-TCRβ, PerCP-CD4, PerCP-CD8α, anti-B220 and PE-sdc1 and analyzed by FACS. CD4 and CD8 T cells were included in one subset (CD4 and CD8) by simultaneously staining samples with PerCP-conjugated anti-CD4 and PerCP-conjugated anti-CD8 mAbs. This allowed us to compare directly SP (CD4⁺ and CD8⁺) and DN (CD4⁻CD8⁻) TCR⁺ subsets. Frequencies of sdc1⁺ DN T cells in the periphery and gut epithelium of wt (A) and gld (C) mice are shown. Numbers indicate the percentage of positive cells in each quadrant. Sdc1⁺ cells in the upper right quadrants in the lymph nodes and spleen of 13- and 21- to 24-week-old gld mice were CD4 T cells (not shown). Percentages of sdc1⁺ DN T cells relative to total T cells in the periphery and gut epithelium of wt (B) and gld (D) mice are shown. DN T cells isolated from the gut epithelium expressed B220 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003465#pone-0003465-g002" target="_blank">Fig. 4A</a> and data not shown). Results are expressed as mean±SD from three independent experiments with two to three mice per group. LN, Lymph nodes, SI, small intestine, Li, large intestine.</p

SEB activation of gld CD8 or CD4 T cells in vivo does not lead to their conversion into DN T cells.

<p>C3H-gld mice (14-week-old) or their age-matched C3H-wt mice were immunized i.p. with SEB (100 µg/mouse). Frequencies of SEB-reactive Vβ8⁺ T cells among the CD8 (A), CD4 (B), and DN (C) subsets in peripheral blood were determined on the indicated days. Similar results were obtained in lymph nodes and spleens when the experiment is terminated. Frequencies of Vβ6⁺ T cells were used as negative controls. Results show mean±SD from three mice per group.</p

Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive Immunotherapy

Adoptive immunotherapy (AIT) can mediate durable regression of cancer, but widespread adoption of AIT is limited by the cost and complexity of generating tumor-specific T cells. Here we develop an Enrichment + Expansion strategy using paramagnetic, nanoscale artificial antigen presenting cells (aAPC) to rapidly expand tumor-specific T cells from rare naïve precursors and predicted neo-epitope responses. Nano-aAPC are capable of enriching rare tumor-specific T cells in a magnetic column and subsequently activating them to induce proliferation. Enrichment + Expansion resulted in greater than 1000-fold expansion of both mouse and human tumor-specific T cells in 1 week, with nano-aAPC based enrichment conferring a proliferation advantage during both <i>in vitro</i> culture and after adoptive transfer <i>in vivo</i>. Robust T cell responses were seen not only for shared tumor antigens, but also for computationally predicted neo-epitopes. Streamlining the rapid generation of large numbers of tumor-specific T cells in a cost-effective fashion through Enrichment + Expansion can be a powerful tool for immunotherapy